Trichohyalin is a major differentiation product of the inner root sheath cells of the hair follicle, the medulla of the hair fiber, and is expressed in the epidermis, and certain other tissues. It is thought to function in part as a keratin intermediate filament associated protein in these tissues. Trichohyalin is a substrate for transglutaminases (TGases), which crosslink it into polymers, and for the enzyme peptidylarginine deiminase (PAD) which converts protein-bound arginines to citrullines. We have expressed in bacteria domain 8 (representing about 40%) of human trichohyalin, and used it to study these two postsynthetic modification events. PAD converts 60% of the arginines to citrullines, resulting in the complete loss of its alpha-helical structure. Trichohyalin is used by all three TGases known to be present in the epidermis, but by calculation of kinetic parameters, TGase 3 enzyme uses it most efficiently. The kinetic efficiency of the TGase 3 enzyme is greatly increased following PAD modification. These data suggest that trichohyalin is first modified by PAD before crosslinking. We have synthesized peptides in order to study the unique single-stranded alpha-helical structural properties of trichohyalin. We have explored the regulation of expression of the human trichohyalin gene. The proximal promoter of the gene exists in the first 135 bp above the transcription start site, and consists of an AP1 element which confers keratinocyte specific expression.